Bone healing is complex process involving a number of different factors including soluble factors such as bone morphogenetic proteins (BMPs), signaling / transcription factors, nuclear transcription factors as well as extracellular matrix components. Although the delivery of BMPs as recombinant proteins can induce local bone formation and healing of bone defects, we and others have demonstrated previously that local gene transfer of BMP-2 at the site of critical size femoral and cranial defects in the rabbit and rat resulted in more rapid and efficient bone healing. More recently, we have shown that gene transfer of the LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation. In humans, three different LMP splice variants have been identified, termed LMP-1, LMP-2, and LMP-3. Gene transfer of human LMP-1 and LMP-3 induces expression of genes involved in bone formation including certain bone morphogenetic proteins (BMPs), promotes bone nodule formation in vitro and ectopic bone formation in vivo, facilitates healing of rat segmental and mandibular bone defects critical size defects and can facilitate posterior thoracic and lumbar spine fusion healing. We also have shown that LMP can induce myogenesis, under certain conditions, in culture. We have demonstrated that at least four different regions of LMP-1 and an additional domain in LMP-3 can contribute to osteogenesis. In partiular, a 20 amino acid region in LMP-3, termed Osteoinductive Domain-1 (OD-1), is able to induce mineralization and bone specific gene expression in cell culture and confer induction of ectopic bone formation in vivo. A synthetic OD-1 peptide fused to a protein transduction domain also was able to induce mineralization and bone specific genes. Thus the goals of this proposal are to identify the minimal as well as optimal LMP-1 and LMP-3 domains required for osteogenesis and myogenesis using cell culture assays, to examine the pathways through which the minimal, domains in LMP are able to induce osteogenesis (BMP signaling, RunX2/OSX transcription and/or MAP kinase) and to examine the ability of the LMP-1 and LMP-3-derived domains to induce efficient and appropriate bone formation in vivo following protein-transduction mediated delivery. The successful completion of the proposal experiments will lead to a better understanding of the pathways important for induction of osteogenesis by LMP and will result in clinically relevant approaches to stimulate new bone formation using LMP-derived peptides. PUBLIC HEALTH RELEVANCE: Bone healing is complex process involving a number of different factors. Although the delivery of BMPs as recombinant proteins can induce local bone formation and healing of bone defects, we have demonstrated previously that local gene transfer of BMPs results in more rapid and efficient bone healing. We have shown that gene transfer of the LIM Mineralization Protein (LMP) can induce osteogenesis and bone formation as or more efficiently than BMP-2. The goals of this proposal are to identify the domains in LMP required for osteogenesis, to begin to examine the mechanisms through which LMP is able to induce osteogenesis and to examine the ability of the LMP-based domains to induce bone formation in vivo following gene and protein- transduction mediated delivery. The successful completion of the proposal experiments will lead to a better understanding of the pathways important for induction of osteogenesis by LMP and will result in clinically relevant approaches to stimulate new bone formation using LMP peptides.